1. Field of the Invention
The present invention relates to bioassay screening generally and, more particularly, but not by way of limitation, to a novel system for ultra high throughput bioassay screening.
2. Background Art
High Throughput Screening (HTS) has been in use for at least the past ten years to screen large numbers of potential chemical compounds that may have pharmaceutical efficacy or which may be precursors to pharmaceuticals. A given investigation may involve the screening of on the order of about 10,000 compounds per day. There are three basic areas of HTS: (1) handling the compound library, (2) lead discovery, and (3) lead optimization. Handling the compound library is an essential element of the other two. Lead discovery and lead optimization tend to overlap. The objective of lead discovery is to develop “hits” or what appear to be active compounds in specific areas. Lead optimization is a refinement of these “hits” so as to pass on qualified leads to medicinal chemistry for further development. Without this refinement, medicinal chemistry is swamped and the discovery of more “hits” is negated. The success of HTS has fostered the next step—a tenfold increase in throughput or Ultra High Throughput Screening (UHTS).
The primary objective of UHTS is to achieve more qualified lead compounds. In general terms, UHTS has been described as the ability to screen, in a given investigation, a library of 500,000 compounds against 50 therapeutic targets per year. This equates to 100,000 compounds screened per day. The economics of this number dictates some form of miniaturization to conserve the precious reagents consumed.
Since compound handling is the front end of both lead discovery and optimization, it must be considered first. The long term library storage is necessarily in solid or semi-solid form, for stability reasons. However, for use in screening, the library must be converted to a liquid phase. The most commonly accepted method of such conversion is to weigh out a small aliquot of a compound and solvate it with dimethyl sulfoxide (DMSO). Speed and convenience dictate weighing out typically 10 milligram quantities as the minimum amount. These are then brought into solution form, at, say, 10 millimolar concentration, yielding 5 to 10 ml of solution. This is then subdivided into smaller aliquots of 0.5 ml and stored frozen in sets of 96 deepwell tubes at −20 or −80 degrees Centigrade as an archive library.
Several areas of concern arise in going from the archive library to the usable form for the assay. First is the concentration—many assays are tested at 10−5 or 10−6 concentrations. The majority of assays cannot tolerate much more than 1% DMSO. Thus, a dilution from the archive library is required. However, some compounds, while soluble in 100% DMSO, are not soluble in lesser percentages. It is desirable to make the compound dilution in the final assay volume and not in a previous dilution step. Another concern is protecting the stability or validity of the archive compound. Freezing it lengthens its shelf life. But to access the compound, it must be thawed to remove an aliquot. Each time a freeze-thaw cycle occurs, there is the potential for moisture to degrade the compound. Thus, it is desirable to minimize these cycles. The real problem is how to transfer 100,000 discreet samples per. day from the archive library to the assay, keeping the above constraints in mind.
Since the libraries may contain upwards of 500,000 discreet compounds, a means is required to both aspirate multiple samples from the compound source and dispense multiple aliquots of nanoliter quantities into the assay destination. Since in the majority of biological assays, a concentration of more than 1% DMSO is toxic to the assay results and if an assay is to run at a 5 microliter volume, only 50 nanoliters of DMSO is allowed. The molarity of the compound solution in DMSO is adjusted so that 50 nanoliters of the compound solution also provides the desired concentration of compound to the assay.
Small individual piezo electric pumps have been utilized for the purpose of aspirating and dispensing these small quantities of liquids. The common method is for the piezo to squeeze an individual glass capillary to create a pressure wave to dispense liquid from within the capillary. Reversing the action will cause the capillary to aspirate liquid. The individual piezo pumps are costly to manufacturer, as are the electronics to drive them. In the pharmaceutical application, described above, it is necessary to rinse the flow passage with a suitable solvent, normally DMSO, to prevent sample-to-sample carry over. Due to the small displacement volume of the piezo pump device a considerable number of cycles or shots is required to pass a suitable quantity of wash fluid. The wash fluid must then be cleared from the pump so as not to dilute the next sample.
In such pharmaceutical research, due to the high numbers to be processed, the samples to be aspirated and dispensed are on very close centers, typically 4.5 mm, or 2.25 mm, or smaller. This places a severe limit on the size of the dispensing device. Due to the small quantities involved, more efficient liquid movement is obtained if the device causing fluid motion is close to the outlet orifice. Otherwise, the energy of the shockwave causing displacement is absorbed by the liquid in the pathway. This results in less velocity at the orifice. If the stream does not have sufficient velocity and kinetic energy at the orifice, it does not overcome the surface tension there and the form of delivery is as liquid drops; however, an ejected stream is desired especially when dispensing. This permits non-contact dispensing. The dispensing tip is not contaminated with other fluids—only the fluid being dispensed.
Accordingly, it is a principal object of the present invention to provide method and means for ultra high throughput screening.
It is a further object of the invention to provide such method and means that are economically implemented.
It is an additional object of the invention to provide such method and means that permit the economical simultaneous aspirating and dispenses of a large number of very small volumes of liquid.
It is another object of the invention to provide such method and means that provide for the compact storage of large numbers of chemical compounds.
It is yet a further object of the invention to provide liquid transfer method and means that employs a single piezoelectric crystal to simultaneously effect the aspiration or dispensing of a large number of liquid samples.
Other objects of the present invention, as well as particular features, elements, and advantages thereof, will be elucidated in, or be apparent from, the following description and the accompanying drawing figures.